Functionalized silicon compounds and methods for their synthesis and use

ABSTRACT

Provided are functionalized silicon compounds and methods for their synthesis and use. The functionalized silicon compounds include at least one activated silicon group and at least one derivatizable functional group. Exemplary derivatizable functional groups include hydroxyl, amino, carboxyl and thiol, as well as modified forms thereof, such as activated or protected forms. The functionalized silicon compounds may be covalently attached to surfaces to form functionalized surfaces which may be used in a wide range of different applications. In one embodiment, the silicon compounds are attached to the surface of a substrate comprising silica, such as a glass substrate, to provide a functionalized surface on the substrate, to which molecules, including polypeptides and nucleic acids, may be attached. In one embodiment, after covalent attachment of a functionalized silicon compound to the surface of a solid silica substrate to form a functionalized coating on the substrate, an array of nucleic acids may be covalently attached to the substrate. Thus, the method permits the formation of high density arrays of nucleic acids immobilized on a substrate, which may be used, for example, in conducting high volume nucleic acid hybridization assays.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/282,979filed Oct. 29, 2002, now U.S. Pat. No. 6,743,882 issued on Jun. 1, 2004,which is a divisional of U.S. application Ser. No. 09/418,044 filed onOct. 13, 1999, now U.S. Pat. No. 6,486,286 issued on Nov. 26, 2002,which is a continuation-in-part of U.S. application Ser. No. 09/172,190filed Oct. 13, 1998, now U.S. Pat. No. 6,262,216 issued Jul. 17, 2001;the disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

This application relates to silicon compounds, methods of making siliconcompounds, and methods for use of silicon compounds as silylating agentsin the treatment of surfaces, such as glass.

BACKGROUND ART

Silylating agents have been developed in the art which react with andcoat surfaces, such as silica surfaces. For example, silylating agentsfor use in modifying silica used in high performance chromatographypackings have been developed. Monofunctional silylating agents have beenused to form monolayer surface coatings, while di- and tri-functionalsilylating agents have been used to form polymerized coatings on silicasurfaces. Many silylating agents, however, produce coatings withundesirable properties including instability to hydrolysis and theinadequate ability to mask the silica surface which may contain residualacidic silanols.

Silylating agents have been developed for the silylation of solidsubstrates, such as glass substrates, that include functional groupsthat may be derivatized by further covalent reaction. The silylatingagents have been immobilized on the surface of substrates, such asglass, and used to prepare high density immobilized oligonucleotideprobe arrays. For example,N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide (PCR Inc.,Gainesville, Fla. and Gelest, Tullytown, Pa.) has been used to silylatea glass substrate prior to photochemical synthesis of arrays ofoligonucleotides on the substrate, as described in McGall et al., J. Am.Chem. Soc., 119:5081–5090 (1997), the disclosure of which isincorporated herein by reference.

Hydroxyalkylsilyl compounds that have been used to preparehydroxyalkylated substances, such as glass substrates.N,N-bis(hydroxyethyl) aminopropyl-triethoxysilane has been used to treatglass substrates to permit the synthesis of high-density oligonucleotidearrays. McGall et al., Proc. Natl. Acad. Sci., 93:13555–13560 (1996);and Pease et al., Proc. Natl. Acad. Sci., 91:5022–5026 (1994), thedisclosures of which are incorporated herein.Acetoxypropyl-triethoxysilane has been used to treat glass substrates toprepare them for oligonucleotide array synthesis, as described in PCT WO97/39151, the disclosure of which is incorporated herein. 3-Glycidoxypropyltrimethoxysilane has been used to treat a glass support to providea linker for the synthesis of oligonucleotides. EP Patent ApplicationNo. 89 120696.3.

Methods have been developed in the art for stabilizing surface bondedsilicon compounds. The use of sterically hindered silylating agents isdescribed in Kirkland et al., Anal. Chem. 61: 2–11 (1989); and Schneideret al., Synthesis, 1027–1031 (1990). However, the use of these surfacebonded silylating agents is disadvantageous, because they typicallyrequire very forcing conditions to achieve bonding to the glass, sincetheir hindered nature makes them less reactive with the substrate.

It is an object of the invention to provide functionalized siliconcompounds that are provided with derivatizable functional groups, thatcan be used to form functionalized coatings on materials, such as glass.It is a further object of the invention to provide functionalizedsilicon compounds that can be used to form coatings on materials thatare stable under the conditions of use.

DISCLOSURE OF THE INVENTION

Provided are functionalized silicon compounds and methods for their use.The functionalized silicon compounds include an activated silicon groupand a derivatizable functional group. Exemplary derivatizable functionalgroups include hydroxyl, amino, carboxyl and thiol, as well as modifiedforms thereof, such as activated or protected forms. The functionalizedsilicon compounds may be covalently attached to surfaces to formfunctionalized surfaces which may be used in a wide range of differentapplications. In one embodiment, the silicon compounds are attached tothe surface of a substrate comprising silica, such as a glass substrate,to provide a functionalized surface on the silica containing substrate,to which molecules, including polypeptides and nucleic acids, may beattached. In one preferred embodiment, after covalent attachment of afunctionalized silicon compound to the surface of a solid silicasubstrate to form a functionalized coating on the substrate, an array ofnucleic acids may be covalently attached to the substrate. Thus, themethod permits the formation of high density arrays of nucleic acidsimmobilized on a substrate, which may be used in conducting high volumenucleic acid hybridization assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the functionalized silicon compounds VIand VII and compounds of Formula 6a.

FIG. 2 shows the structure of the functionalized silicon compound VIII.

FIG. 3 shows schemes for the synthesis of compounds IX and X.

FIG. 4 is a scheme showing the synthesis of compounds of Formula 5 or 6.

FIG. 5 show schemes for the synthesis of compounds XII, XIII andcompounds of Formula 8.

FIG. 6 shows schemes for the synthesis of compounds XV, VI and compoundsof Formula 9.

FIG. 7 shows schemes showing the synthesis of compounds of Formula 10 or11.

FIG. 8 shows schemes showing the synthesis of compounds of Formula 12 or13.

FIG. 9 is a scheme of the synthesis of compounds of Formula 16b.

FIG. 10 illustrates the structure of compounds of Formula 14 and 15.

FIG. 11 is a graph of stability of silicon compound bonded phases vs.time.

FIG. 12 is a graph of hybridization fluorescence intensity vs. silane.

FIG. 13 is a scheme showing the synthesis of silicon compounds XVIa–e.

FIG. 14 is a scheme showing the synthesis of silicon compounds XVIIa–f.

FIG. 15 shows the structure of compounds of the general Formulas 17–21.

FIG. 16 shows the structure of some exemplary silicon compounds.

FIG. 17 shows another embodiment of exemplary silicon compounds XXI andXXII.

FIG. 18 shows the structure of exemplary silicon compounds XXIII, XXVand XXVI.

FIG. 19 shows the structure of exemplary silicon compounds XXIX and XXX.

FIG. 20 shows the structure of exemplary silicon compounds XXIa–b,XXIIa–b and XXIIIa–b.

FIG. 21 is a scheme showing the synthesis of silicon compounds XIX andXX.

FIG. 22 is a scheme showing the synthesis of silicon compounds XXI andXXII.

FIG. 23 is a scheme showing the synthesis of silicon compound XXIII.

FIG. 24 is a scheme showing the synthesis of silicon compound XXIV.

FIG. 25 is a scheme showing the synthesis of silicon compound XXVIII.

FIG. 26 is a scheme showing the synthesis of silicon compounds XXVI andXXV.

FIG. 27 is a scheme showing the synthesis of silicon compounds XXIX andXXX.

FIG. 28 is a scheme showing the synthesis of silicon compounds XXXIa–band XXXIIIa–b.

FIG. 29 is a scheme showing the synthesis of silicon compounds XXXIIa–b.

FIG. 30 is a graph of normalized intensity vs. silane for siliconcompounds bound to a solid substrate.

FIG. 31 is a graph of normalized hybridization fluorescence intensityvs. silane.

MODES FOR CARRYING OUT THE INVENTION

Functionalized silicon compounds are provided, as well as methods fortheir synthesis and use. The functionalized silicon compounds may beused to form functionalized coatings on a variety of surfaces such asthe surfaces of glass substrates.

Functionalized Silicon Compounds

A variety of functionalized silicon compounds, which are availablecommercially, or which may be synthesized as disclosed herein, may beused in the methods disclosed herein to react with surfaces to formfunctionalized surfaces which may be used in a wide range of differentapplications. In one embodiment, the functionalized silicon compoundsare covalently attached to surfaces to produce functionalized surfaceson substrates. For example, the silicon compounds may be attached to thesurfaces of glass substrates, to provide a functionalized surface towhich molecules, including polypeptides and nucleic acids, may beattached.

As used herein, the term “silicon compound” refers to a compoundcomprising a silicon atom. In a preferred embodiment, the siliconcompound is a silylating agent comprising an activated silicon group,wherein the activated silicon group comprises a silicon atom covalentlylinked to at least one reactive group, such as an alkoxy or halide, suchthat the silicon group is capable of reacting with a functional group,for example on a surface of a substrate, to form a covalent bond withthe surface. For example, the activated silicon group on the siliconcompound can react with the surface of a silica substrate comprisingsurface Si—OH groups to create siloxane bonds between the siliconcompound and the silica substrate. Exemplary activated silicon groupsinclude —Si(OMe)₃; —SiMe(OMe)₂; —SiMeCl₂; SiMe(OEt)₂; SiCl₃ and—Si(OEt)₃.

As used herein, the term “functionalized silicon compound” refers to asilicon compound comprising a silicon atom and a derivatizablefunctional group. In a preferred embodiment, the functionalized siliconcompound is a functionalized silylating agent and includes an activatedsilicon group and a derivatizable functional group. As used herein, theterm “derivatizable functional group” refers to a functional group thatis capable of reacting to permit the formation of a covalent bondbetween the silicon compound and another substance, such as a polymer.Exemplary derivatizable functional groups include hydroxyl, amino,carboxy, thiol, and amide, as well as modified forms thereof, such asactivated or protected forms. Derivatizable functional groups alsoinclude substitutable leaving groups such as halo or sulfonate. In onepreferred embodiment, the derivatizable functional group is a group,such as a hydroxyl group, that is capable of reacting with activatednucleotides to permit nucleic acid synthesis. For example, thefunctionalized silicon compound may be covalently attached to thesurface of a substrate, such as glass, and then derivatizable hydroxylgroups on the silicon compound may be reacted with an activatedphosphate group on a protected nucleotide phosphoramidite orH-phosphonate, and then stepwise addition of further protectednucleotide phosphoramidites or H-phosphonates can result in theformation of a nucleic acid covalently attached to the support. Thenucleic acids also may be attached to the derivatizable group via alinker. In a further embodiment, arrays of nucleic acids may be formedcovalently attached to the substrate which are useful in conductingnucleic acid hybridization assays.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides, that comprise purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the polynucleotide cancomprise sugars and phosphate groups, as may typically be found in RNAor DNA, or modified or substituted sugar or phosphate groups. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. The sequence of nucleotides may beinterrupted by non-nucleotide components.

The functionalized silicon compounds used to form coatings on a surfacemay be selected, and obtained commercially, or made synthetically,depending on their properties under the conditions of intended use. Forexample, functionalized silicon compounds may be selected forsilanization of a substrate that are stable after the silylationreaction to hydrolysis.

For example, in one embodiment, the functionalized silicon compounds areused to form a coating on a solid substrate, and include functionalgroups that permit the covalent attachment or synthesis of nucleic acidarrays to the solid substrate, such as glass. The resulting substratesare useful in nucleic acid hybridization assays, which are conducted,for example in aqueous buffers. In one embodiment, preferred are siliconcompounds that produce coatings that are substantially stable tohybridization assay conditions, such as phosphate or TRIS buffer atabout pH 6–9, and at elevated temperatures, for example, about 25–65°C., for about 1 to 72 hours, such that hydrolysis is less than about90%, e.g., less than about 50%, or e.g, less than about 20%, or about10%. The functionalized surfaces on the substrate, formed by covalentattachment of functionalized silicon compounds, advantageously aresubstantially stable to provide a support for biomolecule arraysynthesis and to be used under rigorous assay conditions, such asnucleic acid hybridization assay conditions.

The functionalized silicon compound in one embodiment includes at leastone activated silicon group and at least one derivatizable functionalgroup. In one embodiment, the functionalized silicon compound includesat least one activated silicon group and a plurality of derivatizablefunctional groups, for example, 2, 3, 4 or more derivatizable functionalgroups. In another embodiment, the functionalized silicon compoundincludes at least one derivatizable functional group and a plurality ofactivated silicon groups, for example, 2, 3, 4 or more activated silicongroups. Methods of making the functionalized silicon compounds areprovided as disclosed herein, as well as methods of use of thefunctionalized silicon compounds, including covalent attachment of thesilicon compounds to surfaces of substrates to form functionalizedsurfaces, and further derivation of the surfaces to provide arrays ofnucleic acids for use in assays on the surfaces.

In one embodiment, there is provided a method of functionalizing asurface, the method comprising covalently attaching to the surface afunctionalized silicon compound, wherein the functionalized siliconcompound comprises at least one derivatizable functional group and aplurality of activated silicon groups, for example, 2, 3, 4 or moreactivated silicon groups. The method may further comprise covalentlyattaching a plurality of functionalized silicon compounds to thesurface, and forming an array of nucleic acids covalently attached tothe functionalized silicon compounds on the surface.

Exemplary functionalized silicon compounds include compounds of Formula1 shown below:

wherein R₁ and R₂ are independently a reactive group, such as halide oralkoxy, for example —OCH₃ or —OCH₂CH₃, and R₃ is alkoxy, halide oralkyl; and wherein R₄ is a hydrophobic and/or sterically hindered group.In the functionalized silylating agents of Formula 1, R₄ may be alkyl orhaloalkyl, for example, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —C(CH₃)₃, or—CH(CF₃)₂. A hydrophobic and/or sterically hindered R₄ group, such asisopropyl or isobutyl, may be used to increase the hydrolytic stabilityof the resulting surface layer. Further hydrophobicity may be impartedby the use of a fluorocarbon R₄ group, such as hexafluoroisopropyl((CF₃)₂CH—).

An exemplary compound of Formula 1 is silicon compound I below:

In general, silicon compounds provide uniform and reproducible coatings.Silicon compounds with one derivatizable functional group can provide alower concentration of surface derivatizable functional groups atmaximum coverage of the substrate than the silicon compounds includingmultiple derivatizable functional groups. Silicon compounds with onederivatizable functional group, such as silicon compounds of Formula 1,however, which include a hydrophobic and/or sterically hindered R group,such as isopropyl or isobutyl, are advantageous since the hydrophobic orsterically hindered R group increases the hydrolytic stability of theresulting surface layer.

In one embodiment the functionalized silicon compounds of Formula 2 areprovided:

-   -   In Formula 2, in one embodiment, R₁, R₂ and R₃ are independently        a reactive group, such as alkoxy or halide, for example, —OCH₃,        or —OCH₂CH₃, and wherein, in one embodiment, R₁, R₂ and R₃ are        each —OCH₃. In one embodiment R₁ and R₂ are independently a        reactive group, such as alkoxy or halide, for example —OCH₃ or        —OCH₂CH₃, and R₃ is an alkoxy or halide group or an alkyl group,        such as —CH₃, or substituted alkyl group.    -   In Formula 2, in one embodiment, L₁ and L₂ are independently        alkyl, preferably —(CH₂)_(n)—, wherein n=2 to 10, e.g., 3 to 4,        or e.g., 2–3.    -   In Formula 2, in one embodiment, A₁ is H or a moiety comprising        one or more derivatizable functional groups. In one embodiment,        A₁ is a moiety comprising an amino group or a hydroxyl group,        such as —CH₂CH₂OH. In another embodiment, A₁ is, for example, a        branched hydrocarbon including a plurality of derivatizable        functional groups, such as hydroxyl groups. In one embodiment in        Formula 2, A₁ is:

-   -    wherein p is 1–10, q is 0 or 1, and r is 2–5.

In one embodiment of Formula 2, R₁ and R₂ are independently alkoxy orhalide; R₃ is alkoxy, halide or alkyl; L₁ and L₂ are both —(CH₂)_(n)—,wherein n=2 to 10, e.g., 2 to 3; and A₁ is H or a moiety comprising oneor more derivatizable functional groups.

Exemplary compounds of Formula 2 include compounds II, III and IV below.Other silicon compounds of Formula 2 that may be used to form functionalsurface coatings with enhanced hydrolytic stability include siliconcompounds IX and X, shown in FIG. 3. In compound VIII, thetriethoxysilyl group is shown by way of example, however alternatively,the activated silicon group may be other activated silicon groups ormixtures thereof, such as trimethoxysilyl. In another embodiment, thereis provided a compound of Formula 11, wherein n is, for example, 1 to10, e.g., 1–3, and G is a derivatizable functional group, such ashydroxyl, protected hydroxyl or halide such as Cl or Br, as shown inFIG. 7.

In another embodiment, silicon compounds of Formula 14 in FIG. 10 areprovided wherein, R₁, R₂ are independently a reactive group such asalkoxy, for example —OCH₃ or —OCH₂CH₃, or halide; and R₃ is a reactivegroup such as alkoxy or halide, or optionally alkyl.

Another embodiment of Formula 2 is as follows:

-   -   In one embodiment of Formula 2, R₁, R₂, R₃ are independently a        reactive group, such as alkoxy or halide, for example, —OCH₃, or        —OCH₂CH₃, for example, in one embodiment, R₁, R₂ and R₃ are each        —OCH₃; or in another embodiment, R₁ and R₂ are independently a        reactive group, such as alkoxy or halide, for example —OCH₃ or        —OCH₂CH₃, and R₃ is an alkoxy or halide group or an alkyl group,        such as —CH₃, or substituted alkyl group.    -   In one embodiment of Formula 2, L₁ and L₂ are independently        alkyl, for example, linear or branched alkyl or heteroalkyl,        e.g., C1–C25 alkyl, for example, —(CH₂)_(n)—, wherein n=2 to 10,        e.g., 3 to 4, or e.g., 2–3. For example, L₁ and L₂ may        optionally comprise a heteroalkyl comprising a heteroatom such        as O, S, or N. Each L₁ and L₂ independently comprise one or more        derivatizable groups, e.g., 1–4 derivatizable groups, such as        hydroxyl, amino or amido.    -   In Formula 2, in one embodiment, A₁ is H or a moiety comprising        one or more derivatizable functional groups. In one embodiment,        A₁ is a moiety comprising an amino group or a hydroxyl group,        such as —CH₂CH₂OH. In another embodiment, A₁ is, for example, a        linear or branched alkyl or heteroalkyl group including a        plurality of derivatizable functional groups, for example, 1, 2,        or 3 derivatizable groups. In one embodiment, A₁ may comprise a        linear or branched alkyl or heteroalkyl, wherein one or more        carbon atoms of the alkyl group is functionalized, for example,        to comprise an amide.

Examples of compounds include compounds XVIa–e shown in FIG. 13, andcompounds XVIIIa–f shown in FIG. 14. Other examples include compound XIIin FIG. 5 and compound XV in FIG. 6, as well as compounds XXIX and XXXin FIG. 19.

In a further embodiment, compounds of Formulas 17, 18, 19, and 20 shownin FIG. 15 are provided.

-   -   In one embodiment of the compounds of Formulas 17–20, R₁, R₂, R₃        are independently a reactive group, such as alkoxy or halide,        for example, —OCH₃, or —OCH₂CH₃, for example, in one embodiment,        R₁, R₂ and R₃ are each —OCH₃; or in another embodiment, R₁ and        R₂ are independently a reactive group, such as alkoxy or halide,        for example —OCH₃ or —OCH₂CH₃, and R₃ is an alkoxy or halide        group or an alkyl group, such as —CH₃, or substituted alkyl        group.    -   In one embodiment of the compounds of Formulas 17–20, L₁, L₂,        and L₃ are independently linear or branched alkyl or        heteroalkyl, e.g., C₁₋₂₅ alkyl, for example, —(CH₂)_(n)—,        wherein n=2 to 10, e.g., 3 to 4, or e.g., 2–3. For example, L₁,        L₂, and L₃ may optionally comprise a heteroalkyl comprising a        heteroatom such as O, S, or N. Each L₁, L₂, and L₃ independently        optionally comprise one or more derivatizable groups, e.g., 1–4        derivatizable groups, such as hydroxyl or an amino group.    -   In one embodiment of Formulas 17–20, A₁ and A₂ may independently        comprise H or a moiety comprising one or more derivatizable        functional groups. In one embodiment, A₁ and A₂ are        independently moieties comprising an amino group or a hydroxyl        group, such as —CH₂CH₂OH. In another embodiment, A₁ and A₂ may        independently comprise, for example, a linear or branched alkyl        or heteroalkyl including a plurality of derivatizable functional        groups, for example, 1, 2, or 3 derivatizable groups. In one        embodiment, A₁ and A₂ may independently comprise a linear or        branched alkyl or heteroalkyl, wherein one or more carbon atoms        of the alkyl group is functionalized, for example, to an amide.    -   In one embodiment of the compounds of Formulas 17–20, B₁, and B₂        are independently a branching group, for example alkyl, a        heteroatom, or heteroalkyl, for example a C1–12 alkyl.    -   In one embodiment of the compounds of Formulas 17–20, L₄ is a        direct bond or a linker, for example, C1–12 alkyl or heteroalkyl        optionally comprising one more derivatizable groups.    -   R in one embodiment is H or alkyl or heteroalkyl, for example        C1–12 alkyl, or in another embodiment is acyl, for example        HC(═CH₂)CO— or MeC(═CH₂)CO—.

Examples of compounds of Formula 17 include compounds XIX, XX and XXIVin FIG. 16. Examples of compounds of Formula 19 include compounds XXIand XXII in FIG. 17. Other examples of compounds include compounds XXVIIand XXVIII in FIG. 16.

In one embodiment, compounds of Formula 18 include compounds XXXIa andXXXIb shown in FIG. 20. Compounds of Formula 20 include compounds XXXIIaand XXXIIb shown in FIG. 20.

In another embodiment, the compounds may include a single silicon group,as for example compounds XXXIIIa and XXXIIIb shown in FIG. 20.

In another embodiment, compounds of Formula 21 in FIG. 15 are provided,wherein:

-   -   In one embodiment of Formula 21, R₁, R₂, R₃ are independently a        reactive group, such as alkoxy or halide, for example, —OCH₃, or        —OCH₂CH₃, for example, in one embodiment, R₁, R₂ and R₃ are each        —OCH₃; or in another embodiment, R₁ and R₂ are independently a        reactive group, such as alkoxy or halide, for example —OCH₃ or        —OCH₂CH₃, and R₃ is an alkoxy or halide group or an alkyl group,        such as —CH₃, or substituted alkyl group.    -   In one embodiment of Formula 21, L₁, L₂, L₃, and L₄ are        independently a direct bond, or linear or branched alkyl or        heteroalkyl, e.g., C1–25 alkyl, for example, —(CH₂)_(n)—,        wherein n=2 to 10, e.g., 3 to 4, or e.g., 2–3. For example, L₁,        L₂, L₃ and L₄ may optionally comprise a heteroalkyl comprising a        heteroatom such as O, S, or N. Each L₁, L₂, L₃ and L₄        independently optionally comprise one or more derivatizable        groups, e.g., 1–4 derivatizable groups, such as hydroxyl or an        amino group.    -   In one embodiment, B₁ is a branching group, for example alkyl, a        heteroatom, or heteroalkyl, for example a C1–12 alkyl.    -   In one embodiment, A₁, A₂, A₃, and A₄ may independently comprise        H or a moiety comprising one or more derivatizable functional        groups. In one embodiment, A₁, A₂, A₃, and A₄ are independently        a moiety comprising an amino group or a hydroxyl group, such as        —CH₂CH₂OH. In another embodiment, A₁, A₂, A₃, and A₄ may        independently comprise, for example, an alkyl, such as a linear        or branched alkyl, including a plurality of derivatizable        functional groups, for example, 1, 2 or 3 derivatizable groups.        In one embodiment, A₁, A₂, A₃, and A₄ may independently comprise        a linear or branched alkyl or heteroalkyl, wherein one or more        carbon atoms of the alkyl group is functionalized, for example,        to comprise an amide.

Embodiments of compounds of Formula 21 include compounds XXIII, XXV andXXVI shown in FIG. 18.

In a further embodiment, compounds of Formula 3 are provided:

-   -   In Formula 3, in one embodiment, R₁, R₂ and R₃ are independently        reactive groups, such as alkoxy or halide, for example, —OCH₃,        or —OCH₂CH₃, and wherein, in one embodiment, R₁, R₂ and R₃ are        each —OCH₃. In one embodiment R₁ and R₂ are independently a        reactive group, such as alkoxy or halide, for example —OCH₃ or        —OCH₂CH₃, and R₃ is an alkoxy or halide group or an alkyl group,        such as —CH₃, or substituted alkyl group.    -   In Formula 3, in one embodiment, L₁, L₂, and L₃ are        independently a linker, for example, a straight chain saturated        hydrocarbon, such as —(CH₂)_(n)—, wherein n=1 to 10, or 1 to 5,        or, e.g., 2 to 3.    -   In Formula 3, in one embodiment, A₁ and A₂ are independently H        or moieties comprising one or more derivatizable functional        groups, such as hydroxyl or amino groups, or modified forms        thereof, such as protected forms. In another embodiment, A₁ and        A₂ each comprise a plurality of derivatizable functional groups.        For example, A₁ and A₂ may each comprise a branched moiety        including a plurality of derivatizable functional groups, such        as hydroxyl groups.

In one embodiment of Formula 3, R₁ and R₂ are independently alkoxy orhalide; R₃ is alkoxy, halide or alkyl; L₁, L₂, and L₃ are independently—(CH₂)_(n)—, wherein n is 2–10; and A₁ and A₂ are independently a moietycomprising one or more derivatizable functional groups.

In another embodiment of Formula 3, A₁ is —L₄—G₁ and A₂ is —L₅—G₂; R₁and R₂ are independently alkoxy or halide; R₃ is alkoxy, halide oralkyl; L₁, L₂, L₃, L₄ and L₅ are —(CH₂)_(n)—, wherein n is 1 to 10, forexample 2 to 3; and G₁ and G₂ are independent moiety comprising one ormore derivatizable functional groups. In another embodiment, L₁, L₄, andL₅ are —(CH₂)₂—, L₂ and L₃ are —(CH₂)₃—, and G₂ are —OH.

In another embodiment, silicon compounds of Formula 15 in FIG. 10 areprovided, wherein R₁, R₂ are independently alkoxy, for example —OCH₃ or—OCH₂CH₃, or halide; and R₃ is alkoxy, alkyl, or halide.

In a further embodiment, compounds of Formula 6a in FIG. 1 are provided,wherein n is 1–3, for example 2 or 3. Exemplary functionalized siliconcompounds include compound V below, and compound VI shown in FIG. 1.

Another embodiment is illustrated in FIG. 7, which shows a compound ofFormula 10, wherein n=1 to 10, e.g., 1–3, and G is a derivatizablefunctional group, such as hydroxyl, protected hydroxyl, or halide suchas Cl or Br.

The hydrolytic stability of the silicon compound coating may beincreased by increasing the number of covalent bonds to the surface ofthe support. For example, silicon compounds II–V include two activatedsilicon groups for binding to a support surface, such as glass. Avariety of functionalized silicon compounds including a plurality ofactivated silicon groups and derivatizable functional groups are usefulto form functionalized coatings. A further example is compound VII shownin FIG. 1. Another example is silicon compound VIII shown in FIG. 2,which can form up to three covalent bonds to the surface of a glasssupport. In compound VIII, the triethoxysilyl group is shown by way ofexample, however alternatively, the activated silicon group may be otheractivated silicon groups or mixtures thereof, such as trimethoxysilyl.Similarly, in all of the silicon compounds disclosed herein in which arepresentative activated silicon group, such as trimethoxysilyl, issubstituted on the compound, the compounds in other embodiments also maybe substituted with other activated silicon groups known in the art anddisclosed herein.

The silicon compounds II–VIII having multiple silicon groups enhancepotentially by twice as much, or more, the hydrolytic stability incomparison to silicon compounds comprising only a single silicon group,since they possess more trialkoxysilyl groups that can react, and formbonds with, a surface. The number of silicon groups in the siliconcompound may be modified for different applications, to increase ordecrease the number of bonds to a support such as a glass support.Silicon compounds may be used that form optimally stable surface-bondedfilms on glass via covalent siloxane bonds. Additionally, the number ofderivatizable functional groups may be increased or decreased fordifferent applications, as illustrated by silicon compounds II–VIII.Silicon compounds may be selected for use that provide the desiredoptimum density of surface derivatizable groups, such as hydroxyalkylgroups, for a desired application, such as the synthesis of nucleic acidarrays, or for the optimum stability during use of the array indifferent applications.

Other embodiments of functionalized silicon compounds include compoundXIII shown in FIG. 5.

In another embodiment, polymeric functionalized silicon compounds ofFormula 4 are provided:

-   -   In Formula 4, in one embodiment, x, y and z are independently        1–3 and, in one embodiment, x, y and z are each 2.    -   In Formula 4, in one embodiment, L₁, L₂ and L₃ are independently        linkers, for example, straight chain hydrocarbons, and        preferably —(CH₂)_(m)—, wherein m=1–10, e.g., 2–3.    -   In Formula 4, in one embodiment, at least one of A, B and C is        —SiR₁R₂R₃, wherein R₁ and R₂ are independently a reactive group,        such as alkoxy or halide, for example, —OCH₃, or —OCH₂CH₃ and R₃        is alkoxy, halide or alkyl; and wherein the remainder of A, B        and C are independently moieties comprising one or more        derivatizable functional groups, such as hydroxyl groups, or        amino groups, or modified forms thereof, such as protected        forms, for example —OH or a branched molecule comprising one or        more hydroxyl groups.    -   In Formula 4, in one embodiment, n is, for example, about 10 to        10,000, or, for example, about 1,000 to 10,000.

In one embodiment of Formula 4, B is —SiR₁R₂R₃, wherein R₁, R₂ and R₃are independently alkoxy, halide or alkyl; x, y, and z are independently2–3; L₁, L₂ and L₃ are independently —(CH₂)_(m)—, wherein m is 2–3; Aand C are independently moieties comprising derivatizable functionalgroups; and n is about 10 to 10,000.

In one embodiment of Formula 4, B is —Si(OCH₃)₃; x, y, and z are 2; L₁and L₃ are —(CH₂)₂—; L₂ is —(CH₂)₃—; A and C are moieties comprisingderivatizable functional groups; and n is about 10 to 10,000.

Other embodiments of a polymeric functionalized silicon compound includecompounds of Formula 5 and 6 shown in FIG. 4, wherein m is about 0 to10, e.g., about 1 to 5, and n is about 10 to 10,000. In Formulas 5 and6, R₁ and R₂ are independently a reactive group, such as alkoxy orhalide, for example, —OCH₃ or —OCH₂CH₃, and R₃ is a reactive group, suchas alkoxy or halide, or optionally alkyl, for example —CH₃.

Other embodiments include compounds of Formula 7 and 8, shown in FIG. 5,and Formula 9, shown in FIG. 6, wherein n is about 10 to 10,000. InFormulas 7, 8 and 9, R₁ and R₂ are independently a reactive group, suchas alkoxy or halide, for example, —OCH₃ or —OCH₂CH₃, and R₃ is areactive group such as alkoxy or halide or optionally alkyl, for example—CH₃.

Further embodiments include compounds of Formula 12 and 13, shown inFIG. 8, wherein m is about 10 to 10,000, and n is about 1 to 10, e.g.,about 5 to 10. In Formulas 12 and 13, R₁ and R₂ are independently areactive group, such as alkoxy or halide, for example, —OCH₃ or—OCH₂CH₃, and R₃ is a reactive group, such as alkoxy or halide, oroptionally alkyl, for example —CH₃. In Formula 12, G is a substitutableleaving group, such as hydroxy, protected hydroxy, or halo, such as —Clor —Br.

The use of a polymer permits the formation of stable films on surfaces,such as glass, due to the very large number of siloxane bonds that canbe formed with the surface. The number of alkoxysilicon groups relativeto the number of hydroxyalkyl groups can be selected to provide thedesired density of reactive hydroxyl groups.

Synthesis of Functionalized Silicon Compounds

Functionalized silicon compounds for use in the methods described hereinare available commercially, or may be synthesized from commerciallyavailable starting materials. Commercially available silicon compoundsand a review of silicon compounds is provided in Arkles, Ed., “Silicon,Germanium, Tin and Lead Compounds, Metal Alkoxides, Diketonates andCarboxylates, A Survey of Properties and Chemistry,” Gelest, Inc.,Tullytown, Pa., 1995, the disclosure of which is incorporated herein.Functionalized silicon compounds may be synthesized using methodsavailable in the art of organic chemistry, for example, as described inMarch, Advanced Organic Chemistry, John Wiley & Sons, New York, 1985,and in R. C. Larock, Comprehensive Organic Transformations, Wiley-VCH,New York, 1989.

Methods of synthesizing compounds of Formula 1 are shown in Scheme Ibelow. Commercially available reagents which may be used in syntheses inaccordance with Scheme I include 3-chloro-1-triethoxysilylpropane andethylene oxide (Aldrich®, Milwaukee, Wis.).

A method for the conversion of bis(trimethoxysilylpropyl)amine, XIV,which is commercially available from Gelest, Inc. (Tullytown, Pa.) tocompound II is illustrated below in Scheme II.

A method for the conversion of compound XI,bis[3-trimethoxysilyl)propyl]-ethylenediamine, which is commerciallyavailable from Gelest, Inc., Tullytown, Pa., to compound V is shownbelow in Scheme III.

A method for the synthesis of compound III is shown below in Scheme IV.The reagents shown in Scheme IV are commercially available from Aldrich®(Milwaukee, Wis.).

Reaction schemes for the synthesis of functionalized silicon compoundsIX and X are provided in FIG. 3. Reaction schemes for the synthesis ofcompounds of Formulas 5 and 6 are shown in FIG. 4. Polyethyleneimine isavailable commercially, for example, from Aldrich®. Polyamines ofFormula 5, where R₁, R₂ and R₃ are OMe (trimethoxysilylpropyl modified(polyethyleneimine)), or R₁ is Me and R₂ and R₃ are OMe(dimethoxymethylsilylpropyl modified (polyethylenimine)) are availablefrom Gelest (Tullytown, Pa.). FIG. 9 shows another embodiment of areaction scheme using commercially available reagents, wherein thecompound of Formula 16a is converted to the compound of Formula 16b.

Reaction schemes for the synthesis of compounds XII, XIII, and compoundsof Formula 8 are shown in FIG. 5. Synthesis of the reagent,N,N-bis(2-hydroxyethyl)acrylamide is described in U.S. Pat. No.3,285,886 (1966), the disclosure of which is incorporated herein.

Reaction schemes for the synthesis of functionalized silicon compoundsXV, VI and compounds of Formula 9 are shown in FIG. 6. Use of thereagent N,N-bis(2-hydroxyethyl) -2-chloro-ethylamine is described inOkubo et al., Deutsches Patent 2144759 (1971), the disclosure of whichis incorporated herein.

FIG. 7 illustrates reaction schemes for the synthesis of compounds ofFormulas 10 and 11. the use of the reagent, 4-butanoyl chloride, isdescribed in Njoroge et al., PCT US97/15899 (1988), the disclosure ofwhich is incorporated herein. Other reagents include lactones, such asγ-butyrolactone, δ-valerolactone, and ε-caprolactone (Aldrich®).Compounds XI and XIV are commercially available from Gelest.

FIG. 8 illustrates reaction schemes for compounds of Formulas 12 and 13.In FIG. 8, G is a substitutable leaving group such as halo. Reagents inaddition to those discussed above that may be used in syntheses whichmay be conducted as shown in FIG. 8 include diethanolamine andN,N-bis(2-hydroxyethyl)glycine, which are commercially available, forexample, from Aldrich®.

Further exemplary reaction schemes are shown in FIGS. 13 and 14. FIGS.13 and 14 illustrate examples of methods of synthesis of compounds ofFormula 2, compounds XVIa–e, and XVIIa–f. FIG. 13 illustrates examplesof methods of synthesis where a compound containing a primary aminofunction reacts with two equivalents of(3-glycidoxypropyl)trimethoxysilane (available from Gelest, Inc.,Tullytown, Pa.) to provide tertiary amine containing compoundsXVIa–XVIe. FIG. 14 illustrates examples of methods of synthesis where acompound containing a primary amino function reacts with a carbamoylchloride (produced by reaction of XIV with triphosgene or other phosgenesynthon) to produce the substituted urea compounds XVIIa–XVIIf.

FIG. 21 provides an exemplary reaction scheme for compounds of Formula17. The exemplary compounds XIX and XX are synthesized through thecommon intermediate 4-amino-1,6-heptadiene. This intermediate isprepared from ethylformimidate and methylmagnesium bromide (bothavailable from Aldrich, Milwaukee, Wis.); the preparation is describedin Barbot, F.; Tetrahedron Lett. 1989, 30, 185 and Barber, H. J.; J.Chem. Soc. 1943, 10. Hydrosilylation of 4-amino-1,6-heptadiene followedby reaction with ethylene oxide provides XIX. Acylation of4-amino-1,6-heptadiene with 4-chlorobutyryl chloride (Aldrich,Milwaukee, Wis.) followed by hydrosilylation, and subsequent reaction ofthe resulting disilane with diethanol amine affords XX.

FIG. 22 provides an exemplary reaction scheme for compounds of Formula19. The exemplary compounds XXI and XXII are synthesized through thecommon intermediate 4-allyl-4-amino-1,6-heptadiene. This intermediatecan be prepared, for example, from ethylformimidate and methylmagnesiumbromide (both available from Aldrich, Milwaukee, Wis.); the preparationis described in Barbot, F., Tetrahedron Lett. 1989, 30, 185; and Barber,H. J., J. Chem. Soc. 1943, 101. The intermediate also may be preparedfrom triallylborane as described in Bubnov, Y. N., et al., Russian Chem.Bull. 1996, 45, 2598. Hydrosilylation of 4-ally-4-amino-1,6-heptadienefollowed by reaction with ethylene oxide provides XXI. Acylation of4-allyl-4-amino-1,6-heptadiene with 4-chlorobutyryl chloride (Aldrich,Milwaukee, Wis.) followed by hydrosilylation, and subsequent reaction ofthe resulting trisilane with diethanol amine affords XXII.

FIG. 23 provides an exemplary reaction scheme for compounds of Formula21, including structure XXIII. This compound is prepared by catalytichydrogen reduction of 4,4-dicyano-1,6-bis(triethoxysilyl)heptanefollowed by reaction of the resultant diamine with ethylene oxide. Thedicyano intermediate is prepared, for example, by a malonitrilesynthesis of 4,4-dicyano-1,6-heptadiene followed by hydrosilylation, orby alkylation of malonitrile with 2 equivalents of a3-halo-1-(triethoxysilyl)propane compound.

FIG. 24 provides an exemplary reaction scheme for a compound of Formula17, including structure XXIV. This compound is prepared by catalytichydrogen reduction of 4-cyano-1,6-bis(triethoxysilyl)heptane followed byreaction of the resultant amine with ethylene oxide. The cyanointermediate is prepared starting from a malonitrile synthesis of4,4-dicyano-1,6-heptadiene followed by conversion to4-cyano-1,6-heptadiene by reduction with an organotin reagent asdescribed in Curran, D. P.; et al., Synthesis, 1991, 107.Hydrosilylation of this diene provides4-cyano-1,6-bis(triethoxysilyl)heptane.

FIG. 25 provides an exemplary reaction scheme for compounds of Formula19, for example, structures XXVII and XXVIII. These compounds areprepared by reaction of appropriate primary (XXVIII) or secondary(XXVII) amines with ethyl 1,7-bis(triethoxysilyl)-4-heptanoate. Theester precursor is synthesized starting from diethyl 2,2-diallylmalonate(Aldrich, Milwaukee, Wis.); the diester is decarboxylated following themethod of Beckwith, A. C. J.; et al., J. Chem. Soc., Perkin Trans. II,1975, 1726, to produce ethyl 4-hepta-1,6-dieneoate; the triethoxysilylmoieties are introduced by hydrosilylation.

FIG. 26 provides an exemplary reaction scheme for compounds of Formula21, for example, structures XXV and XXVI. These compounds are preparedby reaction of appropriate primary (XXVI) or secondary (XXV) amines withdiethyl 2,2-bis(3-triethoxysilylpropyl)malonate. The diester precursorcan be synthesized by hydrosilylation of diethyl 2,2-diallylmalonate(Aldrich, Milwaukee, Wis.).

Another embodiment of a synthesis of a compound of Formula 2 is shown inFIG. 27, wherein the synthesis of compounds XXIX and XXX is shown. Thegeneral method illustrated here is to N-alkylate (XXX) or N-acylate(XXIX) bis(3-trimethoxypropyl)amine (compound XI, Gelest, Inc.,Tullytown, Pa.) with a alkyl chain containing an ester of a carboxylicacid that can be subjected to aminolysis with dihydroxyethylamine(Aldrich, Milwaukee, Wis.). The alkylating agent, methyl4-chlorobutyrate, and the acylating agent, methyl4-chloro-4-oxobutyrate, are available from Aldrich (Milwaukee, Wis.).

FIG. 28 provides an exemplary reaction scheme for compounds of Formula18, for example, structures XXXIa and XXXIb. Preparation begins with theformation of 1,6-heptadiene-4-ol from reaction of ethyl formate withallylmagnesium bromide (Aldrich, Milwaukee, Wis.). Reaction of thealcohol with zinc chloride by the method of Reeve, W., J. Org. Chem.1969, 34, 1921 affords a halo diene which after hydrosilylation can beused to alkylate dihydroxyethylamine (Aldrich, Milwaukee, Wis.).N-acylation with acroyl chloride (XXXIa) or methacroyl chloride (XXXIb)(Aldrich, Milwaukee, Wis.) following the procedure of Yokota, M., etal., European patent Application 97309882.5, 1998 affords the desiredcompounds.

FIG. 29 provides an exemplary reaction scheme for compounds of Formula20, including structures XXXIIa and XXXIIb. Preparation begins with theformation of triallylmethanol from reaction of diethyl carbonate withallylmagnesium bromide by the method of Dreyfuss, M. P., J. Org. Chem.1963, 28, 3269. Reaction of the alcohol with zinc chloride by the methodof Reeve, W., J. Org. Chem. 1969, 34, 192 affords a halo diene whichafter hydrosilylation can be used to alkylate dihydroxyethylamine(Aldrich, Milwaukee, Wis.). N-acylation with acroyl chloride (XXXIa) ormethacroyl chloride (XXXIb) (Aldrich, Milwaukee, Wis.) following theprocedure of Yokota, M.; et al., European patent Application 97309882.5,1998 affords the desired compounds.

FIG. 28 provides an exemplary reaction scheme for structures XXXIIIa andXXXIIIb, compounds which contain a single silicon atom. Preparationbegins with the alkylation of dihydroxyethylamine (Aldrich, Milwaukee,Wis.) with an appropriate 3-halo-1-trialkoxylsilylproane reagent,followed by N-acylation with acroyl chloride (XXXIIIa) or methacroylchloride (XXXIIIb) (Aldrich, Milwaukee, Wis.) following the procedure ofYokota, M., et al., European patent Application 97309882.5, 1998 affordsthe desired compounds.

Functionalized silicon compounds within the scope of the invention thatmay be used to form functionalized covalent coatings on surfaces thatare useful in a variety of applications and assays further include aminecompounds such as compound XI, as well as reaction products formedtherefrom as disclosed herein.

Applications

The methods and compositions disclosed herein may be used in a varietyof applications. The functionalized silicon compounds may be covalentlyattached to a variety of materials, to provide derivatizable functionalgroups on the materials. Exemplary materials include materials thatcomprise a functional group that is capable of reacting with theactivated silicon group of the silicon compound. For example, thematerial may comprise a silica material comprising surface silanolscapable of reacting with the activated silicon group to form a siloxanebond between the silicon atom on the silicon compound and the siliconatom on the surface. Thus, the functionalized silicon compounds may beattached to, for example, materials comprising silica, such as glass,chromatography material, and solid supports used for solid phasesynthesis, such as nucleic acid synthesis. The functionalized siliconcompounds further may be attached to materials comprising oxides such astitanium(IV) dioxide and zirconium dioxide, aluminum oxide andindium-tin oxides, as well as nitrides, such as silicon nitride.

Solid substrates which may be coated by the silicon compounds includeany of a variety of fixed organizational support matrices. In someembodiments, the substrate is substantially planar. In some embodiments,the substrate may be physically separated into regions, for example,with trenches, grooves, wells and the like. Examples of substratesinclude slides, beads and solid chips. The solid substrates may be, forexample, biological, nonbiological, organic, inorganic, or a combinationthereof, and may be in forms including particles, strands, gels, sheets,tubing, spheres, containers, capillaries, pads, slices, films, plates,and slides depending upon the intended use.

The functionalized silicon compounds used advantageously may be selectedwith selected properties for a particular application. Functionalizedsilicon compounds may be selected which can form silicon compoundsurface coatings that have good stability to hydrolysis. Functionalizedsilicon compounds may be selected which have a selected reactivity withthe substrate and a selected derivatizable functional group depending onthe intended use.

In one embodiment, the functionalized silicon compounds may becovalently attached to the surface of a solid substrate to provide acoating comprising derivatizable functional groups on the substrate,thus permitting arrays of immobilized oligomers to be covalentlyattached to the substrate via covalent reaction with the derivatizablefunctional groups. The immobilized oligomers, such as polypeptides, ornucleic acids can be used in a variety of binding assays includingbiological binding assays. In one embodiment, high density arrays ofimmobilized nucleic acid probes may be formed on the substrate, and thenone or more target nucleic acids comprising different target sequencesmay be screened for binding to the high density array of nucleic acidprobes comprising a diversity of different potentially complementaryprobe sequences. For example, methods for light-directed synthesis ofDNA arrays on glass substrates is described in McGall et al., J. Am.Chem. Soc., 119:5081–5090 (1997), the disclosure of which isincorporated herein.

Silanation of glass substrates with the silicon compounds describedherein can be conducted, for example by dip-, or spin-application with a1%–10% solution of silicon compound in an aqueous or organic solvent ormixture thereof, for example in 95% EtOH, followed by thermal curing.See, for example, Arkles, Chemtech, 7:766–778 (1997); Leyden, Ed.,“Silanes Surfaces and Interfaces, Chemically Modified Surfaces,” Vol. 1,Gordon & Breach Science, 1986; and Plueddemann, E. P., Ed., “SilaneCoupling Reagents”, Plenum Pub. Corp., 1991, the disclosures of whichare incorporated herein. Methods for screening target molecules forspecific binding to arrays of polymers, such as nucleic acids,immobilized on a solid substrate, are disclosed, for example, in U.S.Pat. No. 5,510,270, the disclosure of which is incorporated herein. Thefabrication of arrays of polymers, such as nucleic acids, on a solidsubstrate, and methods of use of the arrays in different assays, arealso described in: U.S. Pat. Nos. 5,677,195, 5,624,711, 5,599,695,5,445,934, 5,451,683, 5,424,186, 5,412,087, 5,405,783, 5,384,261,5,252,743 and 5,143,854; PCT WO 92/10092; and U.S. application Ser. No.08/388,321, filed Feb. 14, 1995, the disclosures of each of which areincorporated herein. Accessing genetic information using high densityDNA arrays is further described in Chee, Science 274:610–614 (1996), thedisclosure of which is incorporated herein by reference. The combinationof photolithographic and fabrication techniques allows each probesequence to occupy a very small site on the support. The site may be assmall as a few microns or even a small molecule. Such probe arrays maybe of the type known as Very Large Scale Immobilized Polymer Synthesis(VLSIPS®) arrays, as described in U.S. Pat. No. 5,631,734, thedisclosure of which is incorporated herein.

In the embodiment wherein solid phase chemistry, photolabile protectinggroups and photolithography are used to create light directed spatiallyaddressable parallel chemical synthesis of a large array ofpolynucleotides on the substrate, as described in U.S. Pat. No.5,527,681, the disclosure of which is incorporated herein, computertools may be used for forming arrays. For example, a computer system maybe used to select nucleic acid or other polymer probes on the substrate,and design the layout of the array as described in U.S. Pat. No.5,571,639, the disclosure of which is incorporated herein.

Substrates having a surface to which arrays of polynucleotides areattached are referred to herein as “biological chips”. The substrate maybe, for example, silicon or glass, and can have the thickness of amicroscope slide or glass cover slip. Substrates that are transparent tolight are useful when the assay involves optical detection, asdescribed, e.g., in U.S. Pat. No. 5,545,531, the disclosure of which isincorporated herein. Other substrates include Langmuir Blodgett film,germanium, (poly)tetrafluorethylene, polystyrene,(poly)vinylidenedifluoride, polycarbonate, gallium arsenide, galliumphosphide, silicon oxide, silicon nitride, and combinations thereof. Inone embodiment, the substrate is a flat glass or single crystal siliconsurface with relief features less than about 10 Angstroms.

The surfaces on the solid substrates will usually, but not always, becomposed of the same material as the substrate. Thus, the surface maycomprise any number of materials, including polymers, plastics, resins,polysaccharides, silica or silica based materials, carbon, metals,inorganic glasses, membranes, or any of the above-listed substratematerials. Preferably, the surface will contain reactive groups, such ascarboxyl, amino, and hydroxyl. In one embodiment, the surface isoptically transparent and will have surface Si-OH functionalities suchas are found on silica surfaces.

In the embodiment wherein arrays of nucleic acids are immobilized on asurface, the number of nucleic acid sequences may be selected fordifferent applications, and may be, for example, about 100 or more, or,e.g., in some embodiments, more than 10⁵ or 10⁸. In one embodiment, thesurface comprises at least 100 probe nucleic acids each preferablyhaving a different sequence, each probe contained in an area of lessthan about 0.1 cm², or, for example, between about 1 μm² and 10,000 μm²,and each probe nucleic acid having a defined sequence and location onthe surface. In one embodiment, at least 1,000 different nucleic acidsare provided on the surface, wherein each nucleic acid is containedwithin an area less than about 10⁻³ cm², as described, for example, inU.S. Pat. No. 5,510,270, the disclosure of which is incorporated herein.

Arrays of nucleic acids for use in gene expression monitoring aredescribed in PCT WO 97/10365, the disclosure of which is incorporatedherein. In one embodiment, arrays of nucleic acid probes are immobilizedon a surface, wherein the array comprises more than 100 differentnucleic acids and wherein each different nucleic acid is localized in apredetermined area of the surface, and the density of the differentnucleic acids is greater than about 60 different nucleic acids per 1cm².

Arrays of nucleic acids immobilized on a surface which may be used alsoare described in detail in U.S. Pat. No. 5,744,305, the disclosure ofwhich is incorporated herein. As disclosed therein, on a substrate,nucleic acids with different sequences are immobilized each in apredefined area on a surface. For example, 10, 50, 60, 100, 10³, 10⁴,10⁵, 10⁶, 10⁷, or 10⁸ different monomer sequences may be provided on thesubstrate. The nucleic acids of a particular sequence are providedwithin a predefined region of a substrate, having a surface area, forexample, of about 1 cm² to 10⁻¹⁰ cm². In some embodiments, the regionshave areas of less than about 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹, or 10⁻¹⁰ cm². For example, in one embodiment, there isprovided a planar, non-porous support having at least a first surface,and a plurality of different nucleic acids attached to the first surfaceat a density exceeding about 400 different nucleic acids/cm², whereineach of the different nucleic acids is attached to the surface of thesolid support in a different predefined region, has a differentdeterminable sequence, and is, for example, at least 4 nucleotides inlength. The nucleic acids may be, for example, about 4 to 20 nucleotidesin length. The number of different nucleic acids may be, for example,1000 or more. In the embodiment where polynucleotides of a knownchemical sequence are synthesized at known locations on a substrate, andbinding of a complementary nucleotide is detected, and wherein afluorescent label is detected, detection may be implemented by directinglight to relatively small and precisely known locations on thesubstrate. For example, the substrate is placed in a microscopedetection apparatus for identification of locations where binding takesplace. The microscope detection apparatus includes a monochromatic orpolychromatic light source for directing light at the substrate, meansfor detecting fluoresced light from the substrate, and means fordetermining a location of the fluoresced light. The means for detectinglight fluoresced on the substrate may in some embodiments include aphoton counter. The means for determining a location of the fluorescedlight may include an x/y translation table for the substrate.Translation of the substrate and data collection are recorded andmanaged by an appropriately programmed digital computer, as described inU.S. Pat. No. 5,510,270, the disclosure of which is incorporated herein.

Devices for concurrently processing multiple biological chip assays maybe used as described in U.S. Pat. No. 5,545,531, the disclosure of whichis incorporated herein. Methods and systems for detecting a labeledmarker on a sample on a solid support, wherein the labeled materialemits radiation at a wavelength that is different from the excitationwavelength, which radiation is collected by collection optics and imagedonto a detector which generates an image of the sample, are disclosed inU.S. Pat. No. 5,578,832, the disclosure of which is incorporated herein.These methods permit a highly sensitive and resolved image to beobtained at high speed. Methods and apparatus for detection offluorescently labeled materials are further described in U.S. Pat. Nos.5,631,734 and 5,324,633, the disclosures of which are incorporatedherein.

The methods and compositions described herein may be used in a range ofapplications including biomedical and genetic research and clinicaldiagnostics. Arrays of polymers such as nucleic acids may be screenedfor specific binding to a target, such as a complementary nucleotide,for example, in screening studies for determination of binding affinityand in diagnostic assays. In one embodiment, sequencing ofpolynucleotides can be conducted, as disclosed in U.S. Pat. No.5,547,839, the disclosure of which is incorporated herein. The nucleicacid arrays may be used in many other applications including detectionof genetic diseases such as cystic fibrosis, diabetes, and acquireddiseases such as cancer, as disclosed in U.S. patent application Ser.No. 08/143,312, the disclosure of which is incorporated herein. Geneticmutations may be detected by sequencing by hydridization. In oneembodiment, genetic markers may be sequenced and mapped using Type-IIsrestriction endonucleases as disclosed in U.S. Pat. No. 5,710,000, thedisclosure of which is incorporated herein.

Other applications include chip based genotyping, species identificationand phenotypic characterization, as described in U.S. patent applicationSer. No. 08/797,812, filed Feb. 7, 1997, and U.S. application Ser. No.08/629,031, filed Apr. 8, 1996, the disclosures of which areincorporated herein.

Gene expression may be monitored by hybridization of large numbers ofmRNAs in parallel using high density arrays of nucleic acids in cells,such as in microorganisms such as yeast, as described in Lockhart etal., Nature Biotechnology, 14:1675–1680 (1996), the disclosure of whichis incorporated herein. Bacterial transcript imaging by hybridization oftotal RNA to nucleic acid arrays may be conducted as described inSaizieu et al., Nature Biotechnology, 16:45–48 (1998), the disclosure ofwhich is incorporated herein.

All publications cited herein are incorporated herein by reference intheir entirety.

The invention will be further understood by the following non-limitingexamples.

EXAMPLES Example 1

Silicon compounds were obtained commercially or synthesized fromcommercially available starting materials. Silicon compoundsN,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, andN-(2-hydroxyethyl)-N-methyl-3-aminopropyltriethoxysilane (compound I)were purchased from Gelest, Inc (Tullytown, Pa.).

Silicon compounds II and V and were prepared as shown in Schemes II andIII. Solutions of the starting materials,bis(trimethoxysilyl-propyl)amine andbis[(3-trimethoxysilyl)propyl]ethylenediamine in methanol (62 wt %, fromGelest, Inc.) were combined with 1.1–2.2 theoretical equivalents ofethylene oxide at room temperature under a dry ice-acetone condenser.The resulting solutions were analyzed by ¹H-NMR, which indicated 95%conversion in some runs to the hydroxyethylated products, and these wereused without further purification. In other runs, a 60–65% conversionwas obtained for compound V and also was used without furtherpurification.

¹H-NMR(CDCl₃) data are provided below:

(II): 0.55–0.70 (br m, 4H), 1.55–1.65 (br m, 4H), 2.40–2.50 (br m, 2H),2.55–2.60 (br m, 4H), 3.50 (s, MeOH), 3.55 (d, 9H), 3.75 (t, 2H);

(V): 0.55–0.70 (br m, 4H), 1.50–1.65 (br m, 4H), 2.40–2.80 (br m, 12H),3.50 (s, MeOH), 3.40–3.60 (m-s, ˜20H), 3.75 (m, ˜3–4H);

Substrates were treated by a silanation procedure as follows. Glasssubstrates (borosilicate float glass, soda lime or fused silica,2″×3″×0.027″, obtained from U.S. Precision Glass (Santa Rosa, Calif.)were cleaned by soaking successively in Nanostrip (Cyantek, Fremont,Calif.) for 15 minutes, 10% aqueous NaOH/70° C. for 3 minutes, and then1% aqueous HCl for 1 minute (rinsing thoroughly with deionized waterafter each step). Substrates were then spin-dried for 5 minutes under astream of nitrogen at 35° C. Silanation was carried out by soaking undergentle agitation in a freshly prepared 1–2% (wt/vol) solution of thesilicon compound in 95:5 ethanol-water for 15 minutes. The substrateswere rinsed thoroughly with 2-propanol, then deionized water, andfinally spin-dried for 5 minutes at 90°–110° C.

The stability of silicon compound bonded phase was evaluated. Thesurface hydroxyalkylsilane sites on the resulting substrates were“stained” with fluorescein in a checkerboard pattern by first coupling aMeNPOC-HEG linker phosphoramidite, image-wise photolysis of the surface,then coupling to the photo-deprotected linker sites a 1:20 mixture offluorescein phosphoramidite and DMT-T phosphoramidite(Amersham-Pharmacia Biotech, Piscataway, N.J.), and then deprotectingthe surface molecules in 1:1 ethylenediamine-ethanol for 4 hr. The stepswere conducted using standard protocols, as described in McGall et al.,J. Am. Chem. Soc., 119: 5081–5090 (1997), the disclosure of which isincorporated herein.

The pattern and intensity of surface fluorescence was imaged with ascanning laser confocal fluorescence microscope, which employedexcitation with a 488 nm argon ion laser beam focused to a 2 micron spotsize at the substrate surface. Emitted light was collected throughconfocal optics with a 530(+15) nm bandpass filter and detected with aPMT equipped with photon counting electronics. Output intensity values(photon counts/second) are proportional to the amount of surface-boundfluorescein, so that relative yields of free hydroxyl groups withindifferent regions of the substrate could be determined by directcomparison of the observed surface fluorescence intensities. Allintensity values were corrected for nonspecific background fluorescence,taken as the surface fluorescence within the non-illuminated regions ofthe substrate.

The relative surface reactive site density was measured. For eachsilicon compound tested, the number of available surface synthesis sitesachieved per unit area was estimated, relative toN,N-bis(2-hydroxyethyl)-3-aminopropyl-triethoxysilane, by comparison ofthe observed initial surface fluorescence intensities of the varioussubstrates immediately after deprotection in ethanolic diaminoethane.

${{Site}\mspace{14mu}{Density}\mspace{14mu}\left( {\%\mspace{14mu}{{rel}.}} \right)} = \frac{{Intensity}\mspace{14mu}\left( {{silicon}\mspace{14mu}{{compound}\mspace{14mu}}^{``}X^{''}} \right) \times 100}{{Intensity}\mspace{14mu}\left( {{silicon}\mspace{14mu}{compound}\mspace{14mu}{II}} \right)}$

To determine the relative stability of the silicon compound coatings,substrates were gently agitated on a rotary shaker at 45° C. in 5xSSPEor 6xSSPE aqueous buffer (BioWhittacker Products, Walkersville, Md.) atpH 7.3. Periodically, the substrates were removed from the buffer andre-scanned to measure the amount of fluorescein remaining bound to thesurface.

The results are shown in FIG. 11. As shown in FIG. 11, more of thefluorescein tag remained bound to the substrate after prolonged exposureto the aqueous buffer in the case of substrates silanated with II or V,than remained bound to the substrates silanated withN,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane or compound I. Thisdemonstrates that the surface bonded phase obtained with siliconcompounds II or V is much more stable towards hydrolysis than thatobtained with N,N-bis(2-hydroxyethyl)-3-aminopropyl-triethoxysilane.

The hybridization performance of silanated substrates was evaluated. Acomparison was made between substrates silanated withN,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and compound V interms of performance under typical hybridization assay conditions. Anucleic acid probe sequence (5′-GTC AAG ATG CTA CCG TTC AG-3′) (SEQ. IDNO. 1) was synthesized photolithographically in a checkerboard arraypattern (400×400 micron features) on the substrates that had beenderivatized with eitherN,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane or silicon compoundV. After deprotection in ethanolic diaminoethane, the arrays werehybridized with a fluorescein-labeled complementary “target” nucleicacid (5′-fluorescein-CTG AAC GGT AGC ATC TTG AC-3′) (SEQ. ID NO. 2) at aconcentration of 250 pM in 6xSSPE buffer (0.9 M NaCl, 60 mM NaH₂PO₄, 6mM EDTA, pH 7.5) for 16 hours at 45° C. After cooling to roomtemperature, the target nucleic acid solution was removed, and the arraywas washed briefly with 6x SSPE buffer and then scanned on a confocalimaging system. The relative amount of bound target was determined fromthe fluorescence signal intensity. The hybridization signal intensitiesobtained with the more stable substrates, derivatized with siliconcompound V, were at least four times higher than those obtained withsubstrates that were derivatized withN,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane. A graph offluorescence signal intensity vs. silane is shown in FIG. 12.

Example 2

Silicon compound XVIb was prepared as shown in FIG. 13. An oven dried 25ml single-neck flask was charged with 1.07 g (1 ml, 0.0066 mol)N,N-bis(2-hydroxyethyl)-1,3-diaminopropane (TCI America, Portland,Oreg.) and 5 ml anhydrous methanol (Aldrich Chemical; Milwaukee, Wis.).To this stirring solution was added 2.92 ml (3.12 g, 0.0132 mol)(3-glycidoxypropyl)trimethoxysilane (Gelest, Inc., Tullytown, Pa.)dropwise over a 30 minute period. The solution was allowed to stir underan Ar atmosphere until ¹H NMR indicated that the triplet signal for the(CH₂)CH₂(NH₂) protons of the starting amine (δ=2.86 ppm) had shiftedcompletely away (41 hours). Molecular ions corresponding to the startingmaterials were absent in the mass spectrum (positive ion mode) which wasdominated by the [M+H]⁺ peak of m/z=635.5.

Example 3

An alternate preparation of silicon compound XVIb was conducted as shownin FIG. 13. An oven dried 25 ml single-neck flask was charged with 1.07g (1 ml, 0.0066 mol) N,N-bis(2-hydroxyethyl)-1,3-diaminopropane (TCIAmerica, Portland, Oreg.) and 5 ml anhydrous methanol (Aldrich Chemical;Milwaukee, Wis.). To this stirring solution was added 2.92 ml (3.12 g,0.0132 mol) (3-glycidoxypropyl)trimethoxysilane (Gelest, Inc.,Tullytown, Pa.) dropwise over a 30 minute period. The solution wasbrought to reflux and allowed to stir under an Ar atmosphere until ¹HNMR indicated that the triplet signal for the (CH₂)CH₂(NH₂) protons ofthe starting amine (δ=2.86 ppm) had shifted completely away (17 hours).Molecular ions corresponding to the starting materials were absent inthe mass spectrum (positive ion mode), an M+H⁺ peak of m/z=635.5 wasobserved but it did not dominate the spectrum as seen in the roomtemperature preparation of Example 2.

Example 4

Silicon compound XVIe was prepared as shown in FIG. 13. An oven dried 25ml single-neck flask was charged with 2.74 g (0.026 mol)tris(hydroxymethyl)-methylamine (Aldrich Chemical; Milwaukee, Wis.), 10ml (10.70 g, 0.0452 mol) (3-glycidoxypropyl)trimethoxysilane (Gelest,Inc., Tullytown, Pa.), and 11 ml anhydrous methanol (Aldrich Chemical;Milwaukee, Wis.). The suspension was allowed to stir under an Aratmosphere. After all the solids dissolved (6 days), ¹³C NMR indicatedthat the singlet signal for the C(CH₂OH)₃ carbon of the starting amine(δ=55.38 ppm) had shifted completely away. ¹³C NMR (δ, ppm): 73.12(CH—OH), 71.03 (C—N), 61.94 (CH₂OH), 50.62 (C—[OC]), 50.06 ([C]—C—O),49.39 (CH₃OSi), 43.85 (N—C—[CH₂OH]₃), 22.34 (C—[CSi]), 4.81 (C—Si). MassSpectrum: M⁺ (m/z 593.4) and [M-CH₃OH]⁺ (m/z 561.5).

Example 5

A solution of 104 g triphosgene (0.102 mol) (Aldrich Chemical;Milwaukee, Wis.) and 1 liter anhydrous THF (Aldrich Chemical; Milwaukee,Wis.) was prepared under Ar in a 2-1 oven-dried flask. To the stirringsolution was slowly added 42.5 ml (30.8 g, 0.304 mol) triethylamine(Aldrich Chemical; Milwaukee, Wis.) and a white precipitate formed. Thestirring suspension was cooled in ice and 100 mlN,N-bis(3-trimethoxysilylpropyl)amine (104 g, 0.304 mol) (compound XIV,Gelest, Inc., Tullytown, Pa.) was added dropwise over an hour. Thereaction mixture was allowed to stir for 21.5 hours then filtered underan Ar blanket and the solids washed with anhydrous THF. The filtrate wasconcentrated under reduced pressure to an orange oil and dried undervacuum to give 116 g of product (95% crude yield). ¹H NMR (δ, ppm): 3.57(s, CH₃OSi), 3.32 (dd, CH₂—NC═O, 4 H), 1.70 (q, [CH₂]—CH₂—[CH₂], 4 H),0.58 (t, CH₂Si, 4 H). ¹³C NMR (δ, ppm): 149.14 (C═O), 53.53 (C—N—C═O),52.25 (C—N—C═O), 50.66 (CH₃OSi), 21.83 (C—C—N), 20.82 (C—C—N), 6.50(C—Si).

Example 6

Silicon compound XVIIb was prepared as shown in FIG. 14. An oven dried25 ml single-neck flask was charged with 0.21 ml (0.15 g, 0.0015 mol)triethylamine (Aldrich Chemical; Milwaukee, Wis.), 0.61 g of a carbamoylchloride prepared as per Example 5 (0.0015 mol), and 5 ml anhydrous THF(Aldrich Chemical; Milwaukee, Wis.). After addition of 0.20 g (0.0015mol) diethanolamine (Aldrich Chemical; Milwaukee, Wis.) a precipitateformed. After 5 days of stirring under Ar at room temperature, themixture was filtered and concentrated to a brown oil. After drying undervacuum, 0.62 g (87% crude yield) of material was obtained. The oil wasdissolved in 0.6 ml methanol and stored. ¹³C NMR (δ, ppm): 166.29 (C═O),60.50 (C—OH), 57.28 (N—C—COH), 46.14 (C—C—N), 21.01 (C—[CSi]), 6.27(C—Si). Mass Spectrum: [M+H]⁺ (m/z 473) and [M—CH₃O]⁺ (m/z 441).

Example 7

Silicon compound XVIIf was prepared as shown in FIG. 14. An oven dried25 ml single-neck flask was charged with 42.4 ml (30.8 g, 0.304 mol)triethylamine (Aldrich Chemical; Milwaukee, Wis.), 116 g of a carbamoylchloride prepared as per Example 5 (0.304 mol), and 500 ml anhydrous THF(Aldrich Chemical; Milwaukee, Wis.). After addition of 1.07 g (1 ml,0.0066 mol) N,N-Bis(2-hydroxyethyl)-1,3-diaminopropane (TCI America,Portland, Oreg.), 500 ml anhydrous CH₂Cl₂ (Aldrich Chemical; Milwaukee,Wis.) was added. The mixture was stirred under Ar at room temperature,during which time an insoluble orange oil formed. After 18 hours, ¹H NMRindicated that the triplet signal for the (CH₂)CH₂(NH₂) protons of thestarting amine (δ=2.86 ppm) had shifted completely away. The mixture wasdecanted and the filtrate concentrated. The concentrate was combinedwith 400 ml anhydrous THF (Aldrich Chemical; Milwaukee, Wis.). Theresultant suspension was filtered, concentrated under reduced pressure,and dried under vacuum to yield 145 g (95% crude yield) of a faintorange oil. The oil was dissolved in 150 ml methanol and stored. MassSpectrum: [M+H]⁺ (m/z 530).

Example 8

Coated substrates with XVIb, XVIe, XVIIb, and XVIIf were prepared by amodified version of the silanation procedure of Example 1. In this case,the silane content of the 95:5 ethanol:water bath was 1–2% by volume ofthe methanol/silane compound solutions described in Examples 2–4 and 6–7and curing was done at 50° C. for 2 minutes.

Example 9

Evaluation of stability of the silicon bonded phase, relative surfacereactive site density, and hybridization performance of substratescoated with XVIb, XVIe, XVIIb, and XVIIf was carried out in a singleexperimental protocol. First, MeNPOC-HEG linker phosphoramidite iscoupled to the surface, then image-wise photolysis is used to patternthe surface into a series of stripes. Two stripes, 400 μm×12800 μm, werestained with fluorescein label to evaluate stability of the siliconbonded phase and relative surface reactive site density. A nucleic acidprobe sequence was synthesized photolithographically into two additionalstripes, 1600 μm×12800 μm. After patterning, the surface is deprotectedin 1:1 ethylenediamine ethanol for 4 hours. The steps were conductedusing standard protocols, as described in McGall et al., J. Am. Chem.Soc., 119:5081–5090 (1997), the disclosure of which is incorporatedherein. The pattern and intensity of surface fluorescence from bothfluorescein labeled patterns and adsorbed fluorescein labeled targetoligonucleotide is imaged with a scanning fluorescence microscope asdescribed in Example 1.

In the assay the surface was imaged after a period of 1 hour exposure to5 nM target in 6x SSPE buffer (0.9 M NaCl, 60 mM NaH₂PO₄, 6 mM EDTA, pH7.5) at 25° C. and again after 16 hours at 45° C. Except as notedherein, the method employed for the assay is the same as described inExample 1. The fluorescence intensity was averaged and evaluated at boththe 1 hour and 16 hour time points. Relative surface reactive sitedensity was determined by normalization of the signal to that obtainedfrom compound I as described in Example 1. FIG. 30 shows the normalizeddata at the 1 and 16 hour time points. The percentage of remainingfluorescence intensity is noted for the 16-hour time point for eachsilane coating.

Hybridization performance of substrates for matched probes isillustrated in FIG. 31, where the 1 hour, 25° C. intensities of theaveraged background corrected fluorescence signals of the matchedsequence stripes are shown. Signals are normalized to 1=intensity ofidentical probes synthesized onN,N-bis(2-hydroxyethyl)-3-trimethoxypropylamine (compound 1) surfaces.

1. A method of functionalizing a surface, the method comprisingcovalently attaching to the surface a functionalized silicon compound ofFormulas 17, 18, 19, or 20:

wherein: R₁ and R₂ are independently alkoxy or halide, and R₃ is alkoxy,halide or alkyl; L ₁, L₂ and L₃ are independently linear or branchedalkyl, or linear or branched heteroalkyl, optionally comprising one ormore derivatizable groups; A₁ and A₂ independently are H or a moietycomprising one or more derivatizable functional groups; B₁ and B₂ areindependently alkyl, a heteroatom, or heteroalkyl; L₄ is a direct bond,alkyl or heteroalkyl optionally comprising one or more derivatizablegroups; and R is H, alkyl, heteroalkyl, or acyl.
 2. The method of claim1, wherein the method comprises covalently attaching a plurality offunctionalized silicon compounds to the surface; and forming an array ofnucleic acids covalently attached to the functionalized siliconcompounds on the surface.
 3. The method of claim 1, wherein thederivatizable groups are hydroxyl groups or modified forms thereof. 4.The method of claim 1, wherein the compound is selected from the groupconsisting of


5. The method of claim 1, wherein the surface is the surface of asubstrate comprising silica.
 6. The method of claim 1, wherein R₃ is asubstituted alkyl group.
 7. The method of claim 1, wherein R₁, R₂, andR₃, are independently, —OCH₃, or —OCH₂CH₃.
 8. The method of claim 7,wherein R₁, R₂ and R₃ are each —OCH₃.
 9. The method of claim 1, whereinR₁, and R₂, are independently, —OCH₃ or —OCH₂CH₃, and R₃ is —CH₃, or asubstituted alkyl group.
 10. The method of claim 1, wherein L₁, L₂, andL₃ are independently, C₁₋₂₅ alkyl; or a heteroalkyl; and thederivatizable groups are hydroxyl or amino.
 11. The method of claim 10,wherein L₁, L₂, and L₃ are independently, —(CH₂)_(n)—, wherein n is aninteger from 2 to
 10. 12. The method of claim 11, wherein L₁, L₂, and L₃are independently, —(CH₂)_(n)—, wherein n is 2, 3, or
 4. 13. The methodof claim 1, wherein A₁ and A₂ independently comprise, a linear orbranched alkyl or linear or branched heteroalkyl, having 1, 2, or 3derivatizable groups.
 14. The method of claim 13, wherein thederivatizable groups are amino, or hydroxyl.
 15. The method of claim 1,wherein A₁ and A₂ are independently —CH₂CH₂OH, or an amide.
 16. Themethod of claim 1, wherein B₁ and B₂ are independently C₁₋₁₂ alkyl,branched C₁₋₁₂ alkyl, a heteroatom, or branched C₁₋₁₂ heteroalkyl. 17.The method of claim 1, wherein L₄ is C₁₋₁₂ alkyl or C₁₋₁₂ heteroalkyloptionally comprising one more derivatizable groups.
 18. The method ofclaim 1, wherein acyl is HC(═CH₂)CO— or MeC(═CH₂)CO—.